EXPRESSION OF PHYTASE IN ASPERGILLUS NIGER

Abstract

Disclosed herein is a method for expressing phytase in a filamentous fungus by using an optimized Escherichia coli phytase gene having a nucleotide sequence as shown in SEQ ID NO. 7 and a signal peptide having a nucleotide sequence as shown in SEQ ID NO. 12.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of genetic engineering, and to the high-efficiency expression of phytase derived from Gram-negative bacteria, particularly Escherichia coli phytase, in filamentous fungi, especially Aspergillus niger.

BACKGROUND

[0002] Phytase, that is, myo-Inositol hexakisphosphate phosphohydrolase, is an orthophosphoric-monoester phosphohydrolase, which catalyzes the hydrolysis of phytic acid to produce lower inositol phosphate derivatives and inorganic phosphoric acid. In some instances, phytic acid can be hydrolyzed into free inositol. Phytic acid is the most abundant in the seeds of crops such as grains, beans and oil crops, and is present in an amount of up to 1% to 3%, accounting for 60% to 80% of the total phosphorus content in the plants. However, the phosphorus in physic acid cannot be directly absorbed and utilized, and needs to be hydrolyzed into inorganic phosphate in the digestive tract. Studies have shown that monogastric animals (for example, pigs, chickens, ducks, and gooses, etc.) have low utilization of phosphorus in phytic acid due to the lack of phytase. Meanwhile, the strong electronegativity of phytic acid causes it to form insoluble salts with a divalent or trivalent cation such as Ca.sup.2+, Zn.sup.2+, and Fe.sup.2 ++, hindering the absorption of minerals in the small intestine. It also forms complexes with proteins, amino acids and fatty acids, affecting their absorption and utilization. Phytic acid also binds to pepsin, chymotrypsin, trypsin, and others to reduce the digestive enzyme activity. Therefore, the addition of phytase to feed for monogastric animals can increase the utilization of phosphorus in the feed, reduce the phosphorus content in the animal excrements, and increase the energy utilization rate of the proteins and the feed.

[0003] Commercial phytase is primarily derived from Aspergillus niger (as described in U.S. Pat No. 5,436,156), Escherichia coli (as described in U.S. Pat. No. 7,432,098), Citrobacter genus (such as the Citrobacter braakii strain described in US 20100261259), and Brucella (such as Buttiauxella sp. described in U.S. Pat. No. 8,143,046) and so on. These phytases have different acid and heat resistances due to their different origins. Nielsen et al. (J Agric Food Chem. 2015, 63(3): 943-50) compared the performances of commercial phytases and showed that Escherichia coli phytase exhibits the best performances. Commercial Escherichia coli phytase products are all expressed in yeasts, such as schizosaccharomyces and Pichia pastoris, and most of the phytase products on the Chinese market are produced by Pichia pastoris. Since phytase is mainly used in the areas of feed and food, and Pichia pastoris needs to use methanol as a carbon source to induce protein expression when a protein is expressed in Pichia pastoris, it is difficult to completely remove methanol as a raw material from commercial phytase, thus causing potential safety hazards. In addition, due to the flammable and explosive nature of methanol, special safety protection is required during transportation and production, which increases the production costs. Moreover, higher requirements are imposed on the production environment, and there are certain potential hazards to the production workers. It is therefore not preferred to use Pichia pastoris to produce food and feed additives. Filamentous fungi are well known as cell factories for producing valuable products (such as enzymes). Among them, Aspergillus niger and Aspergillus oryzae are widely used as expression hosts because of their "Generally Recognized As Safe (GRAS)" characteristics. During the fermentation process, no toxic substances are produced, and the raw materials for fermentation are all cereals and by-products thereof (such as soybean meal, corn syrup, etc.). Therefore, Aspergillus niger and Aspergillus oryzae are more favored in the industry for the production of enzymes. It has been found through studies that the use of Aspergillus niger to express phytase derived from Gram-negative bacteria is difficult. For example, US 20100261259 describes the use of Aspergillus oryzae, Aspergillus niger and yeast to express Citrobacter braakii (a Gram-negative bacterium) phytase. The results show that Aspergillus oryzae and yeast can well secrete and express the phytase, while Aspergillus niger can hardly express the phytase. As described above, the phytase derived from the Gram-negative bacterium (Escherichia coli) and its mutant have better characteristics, and therefore the expression in Aspergillus niger that is GRAS is expected.

SUMMARY

[0004] An object of the present invention is to provide a signal peptide which enhances the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus.

[0005] Another object of the present invention is to provide a codon-optimized gene encoding the Escherichia coli phytase or a mutant thereof.

[0006] Another object of the present invention is to provide a codon-optimized DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof.

[0007] A further object of the present invention is to provide a method for enhancing the secretory expression of the Escherichia coli phytase or a mutant thereof in a filamentous fungus.

[0008] The objects of the present invention can be accomplished through the following technical solutions:

[0009] In one aspect, the present invention provides a signal peptide for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus, where the signal peptide is selected from the Aspergillus oryzae TAKA amylase signal peptide having an amino acid sequence as shown in SEQ ID NO. 13.

[0010] In one embodiment of the present invention, the nucleotide sequence of the signal peptide is as shown in SEQ ID NO. 12.

[0011] In one embodiment of the present invention, the filamentous fungus is selected from Aspergillus niger.

[0012] In one embodiment of the present invention, the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 4.

[0013] In another embodiment of the present invention, the mutant of the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

[0014] In another aspect, the present invention provides a codon-optimized gene encoding Escherichia coli phytase or a mutant thereof, which has a nucleotide sequence as shown in SEQ ID NO. 7; or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 7, and encodes a protein having the phytase activity.

[0015] In one embodiment of the present invention, the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 4.

[0016] In another embodiment of the present invention, the mutant of the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

[0017] In still another aspect, the present invention provides a codon-optimized DNA sequence encoding Escherichia coli phytase or a mutant mature peptide thereof, which has a nucleotide sequence as shown in SEQ ID NO. 8, or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 8, and encodes a protein having the phytase activity.

[0018] In one embodiment of the present invention, the codon-optimized DNA sequence encoding Escherichia coli phytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 14.

[0019] In another embodiment of the present invention, the codon-optimized DNA sequence encoding Escherichia coli phytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 16.

[0020] In still another aspect, the present invention provides a method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus. The method comprises linking the Aspergillus oryzae TAKA amylase signal peptide to a DNA sequence encoding Escherichia coli phytase or a mutant mature peptide thereof, and inserting an expression cassette comprising the sequence into a filamentous fungus for expression, where the nucleotide sequence of the Aspergillus oryzae TAKA amylase is as shown in SEQ ID NO. 12.

[0021] In one embodiment of the present invention, the amino acid sequence of the Aspergillus oryzae TAKA amylase is as shown in SEQ ID NO. 13.

[0022] In one embodiment of the present invention, the filamentous fungus is selected from Aspergillus niger.

[0023] In one embodiment of the invention, the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 4, and the mutant of the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

[0024] In one embodiment of the present invention, the DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof is not codon optimized.

[0025] In another embodiment of the present invention, the DNA sequence encoding Escherichia coli phytase or a mutant mature peptide thereof is codon optimized.

[0026] In another embodiment of the present invention, the codon-optimized DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof has a nucleotide sequence as shown in SEQ ID NO. 8; or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 8, and encodes a protein having the phytase activity.

[0027] In a preferred embodiment of the present invention, the codon-optimized DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 14.

[0028] In a preferred embodiment of the present invention, the codon-optimized DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 16.

[0029] An expression cassette, a recombinant expression vector, a recombinant strain, a transgenic cell line or a recombinant strain comprising the gene encoding the Escherichia coli phytase is also provided.

[0030] To construct an Escherichia coli phytase expression cassette, a specific promoter, terminator, signal peptide sequence, and regulatory sequence are required. e.g., 5' UTR, 3' UTR, and the like.

[0031] The promoter may be an endogenous promoter from Aspergillus niger, such as the glycosylase gene promoter, neutral amylase gene promoter, acid amylase gene promoter, and .alpha.-glucosidase gene promoter, etc. from Aspergillus niger; or an exogenous promoter, such as the neutral amylase gene promoter from Aspergillus oryzae, glycosylase gene promoter from Rhizopus oryzae; or a promoter variant, such as the Aspergillus niger neutral amylase gene promoter variant. In the present invention, the Aspergillus niger glycosylase gene promoter or Aspergillus niger neutral amylase gene promoter variant is preferred.

[0032] A regulatory sequence may be linked to the 3' end of the promoter, for example, a suitable leader sequence (5' UTR), that is, an untranslated region of mRNA important for the translation of the host cell, such as the leader sequence of Aspergillus oryzae neutral amylase and Aspergillus nidulans triose-phosphateisomerase;

[0033] For the secretory expression of a specific protein, a signal peptide sequence is required, and in the present invention, the Aspergillus oryzae TAKA amylase signal peptide is preferred for the Escherichia coli phytase.

[0034] Preferred tetrriinators are obtained from the genes of Aspergillus niger glycosylase, Aspergillus oryzae TAKA amylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase and Fusarium oxysporum trypsin-like protease.

[0035] A specific gene is linked to the promoter, the regulatory sequence, the signal peptide sequence, and the terminator to form an expression cassette. It can be inserted into the genome of Aspergillus niger by conventional methods, and can be randomly inserted into the genome or integrated into one or more loci. Optional loci include gla (glycosylase), amya (neutral amylase), amyb (neutral amylase), aa (acid amylase), agda (alpha glucosidase), and agdb (alpha glucosidase).

[0036] The expression cassette can preferably be linked with one or more selectable markers, which allow(s) for simple selection of cells or strains that have been transformed, transfected, and transduced. The selectable marker is a gene whose product provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hyg (hygromycin phosphofransferose), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulphate adenylyltransferase) and trpC (anthranilate synthase) and their equivalents. Preferred for use in Aspergillus cells are amdS and hyg of Aspergillus nidulans or Aspergillus oryzae.

[0037] The expression cassette is preferably linked to one or more counter-selectable markers (negative selection markers). Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), pyrG (orotidine-5'-phosphate decarboxylase), and hsvTK (herpes simplex virus thymidine kinase).

[0038] A recombinant expression vector is obtained by inserting the Escherichia coli phytase gene or an expression cassette containing the gene into an expression vector.

[0039] A recombinant strain is obtained by introducing the recombinant expression vector into a host strain of interest which is preferably Aspergillus niger.

[0040] Use of the Escherichia coli phytase gene in increasing the expression level of Escherichia coli phytase is provided.

[0041] Use of the expression cassette, the recombinant expression vector, and the recombinant strain in increasing the expression level of Escherichia coli phytase is also provided.

[0042] A method for producing Escherichia coli phytase is provided, which comprises fermenting and culturing the transgenic recombinant strain to obtain Escherichia coli phytase.

[0043] The method for introducing a DNA fragment into Aspergillus niger in the present invention is a conventional method in the art.

[0044] Codon optimization refers to the redesign of genes by using preferred codons instead of low-utilization or rare codons. For a detailed description of codon optimization, see the article of Joshua B. Plotkin and Grzegorz Kudla (Nat Rev Genet. 2011; 12(1): 32-42.). Codon optimization has been widely used in heterologous expression systems.

[0045] Beneficial Effects of the Present Invention:

[0046] The present inventors have found that for the gene encoding Escherichia coli phytase or a mutant thereof, it is preferred to synthesize an artificial gene after codon optimization, and after being linked with the Aspergillus oryzae TAKA amylase signal peptide, the constructed expression cassette is introduced into Aspergillus niger for expression. In this way, a large amount of secreted and expressed phytase can be obtained in the culture supernatant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 is mapping of a pHphtk plasmid.

[0048] FIG. 2 is mapping of a pGla-Phy-Phy plasmid.

[0049] FIG. 3 is mapping of a pGla-Gla-Phy plasmid.

[0050] FIG. 4 is mapping of a pGla-Amy-Phy plasmid.

[0051] FIG. 5 is mapping of a pGla-Phy-PhyOPT plasmid.

[0052] FIG. 6 is mapping of a pGla-Gla-PhyOPT plasmid.

[0053] FIG. 7 is mapping of a pGla-Amy-PhyOPT plasmid.

[0054] FIG. 8 is mapping of a pGla-Amy-PhyM1 plasmid.

[0055] FIG. 9 is mapping of a pGla-Amy-PhyM2 plasmid.

DETAILED DESCRIPTION

EXAMPLE 1

Construction of pHphtk Plasmid

[0056] The plasmid contains the following three parts, and is constructed by Nanjing Kingsray Biotechnology Co., Ltd., and the mapping of the plasmid is shown in FIG. 1.

[0057] (1) a 2305 bp fragment obtained by XbaI-PciI double digestion of pUC57 plasmid;

[0058] (2) a hph gene expression cassette, having a sequence as shown in SEQ ID NO. 18; and

[0059] (3) an HSV-tk expression cassette, having a sequence as shown in SEQ ID NO. 19.

EXAMPLE 2

Construction of Plasmid Integrated with Escherichia coli Phytase Guided by Various Signal Peptides

[0060] An Escherichia coli phytase expression cassette was integrated into the Aspergillus niger glycosylase locus for expression, where the glycosylase promoter and the glycosylase terminator were used. pGla-Phy-Phy, pGla-Gla-Phy, and pGla-Amy-Phy plasmids were constructed respectively. Various signal peptide sequences including the Escherichia coli phytase signal peptide (SEQ ID NO. 5), Aspergillus niger glycosylase signal peptide (SEQ ID NO. 10), and Aspergillus oryzae TAKA amylase signal peptide (SEQ ID NO. 12) were respectively linked to the wide-type Escherichia coli phytase mature peptide encoding DNA sequence Phy (SEQ ID NO. 3), and then used to replace the Aspergillus niger glycosylase gene. The phytase mature peptide encoding DNA sequence Phy (SEQ ID NO.3) derived from Escherichia coli ATCC 8739 was synthesized by Nanjing Kingsray Biotech Co., Ltd., the Phy signal peptide DNA sequence was synthesized by Nanjing Kingsray Biotech Co., Ltd., the Aspergillus niger glycosylase signal peptide (SEQ ID NO.10) and the Aspergillus oryzae TAKA amylase signal peptide (SEQ ID NO.12) were introduced onto the Phy sequence by PCR using primers. The integrated plasmid was constructed as follows. The pHphtk plasmid was linearized by vector-F and vector-R primers. The genome of Aspergillus niger (from China Center of Industrial Culture Collection under Accession No. CICC2462) was used as a template, and the Gla-5'-F and Gla-5'-R and the Gla-3'-F and Gla-3'-R were respectively used to amplify the 5' and 3' flanking sequences of the glycosylase gene, where each fragment was 2000 bp long. The wild-type Escherichia coli phytase sequence Phy (SEQ ID NO. 1) was amplified using Phy-Phy-F and Phy-Phy-R. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Phy fragment were recombined by Gibson Assembly.RTM. Master Mix Kit (E2611, New England Biolabs) to obtain an integrated plasmid pGla-PepWT, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 2. Gla-Phy-F and Phy-Phy-R were used as primers, and the Phy fragment (SEQ ID NO. 1) was used as a template to obtain a Gla-Phy fragment by PCR amplification. In this fragment, the Aspergillus niger glycosylase signal peptide sequence was introduced. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Gla-Phy fragment were recombined by Gibson Assembly.RTM. Master Mix Kit to obtain an integrated plasmid pGla-Gla-Phy, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 3. Amylase-Phy-F and Phy-Phy-R were used as primers and the Phy fragment (SEQ ID NO. 1) was used as a template to obtain an Amylase-Phy fragment by PCR amplification. In this fragment, the Aspergillus oryzae TAKA amylase signal peptide sequence was introduced. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Amylase-Phy fragment were recombined by Gibson Assembly.RTM. Master Mix Kit to obtain an integrated plasmid pGla-Amy-Phy, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 4. The 2 kb 5'-terminal flanking DNA sequence of the glycosylase gene is shown in SEQ ID NO. 20, and the 2 kb 3'-terminal flanking DNA sequence is shown in SEQ ID NO. 21. Phy-Phy, Gla-Phy and Amy-Phy are as shown in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, respectively.

[0061] Related primer sequences are listed below:

TABLE-US-00001 Primer name Sequence (5'.fwdarw.3') vector-F gtacagtgaccggtgactctttctggcatg vector-R gatgcattcgcgaggtaccgagctc Gla-5'-F aattcgagctcggtacctcgcgaatgcatcctacca atgctctcgaggattgcctgaacattgacattcggc Gla-5'-R tgctgaggtgtaatgatgctggg Gla-3'-F acaatcaatccatttcgctatagttaaaggatg Gla-3'-R catgccagaaagagtcaccggtcactgtacatggc caatgtggtagccgttatcag Phy-Phy-F cttcatccccagcatcattacacctcagcaatgtc agatatgaaaagcggaaacatatc Phy-Phy-R cattaactatagcgaaatggattgattgtttacaa actgcacgccggtatgc Gla-Phy-F cttcatccccagcatcattacacctcagcaatgtc gttccgatctctactcgccctgagcggcctcgtct gcacagggttggcaaatgtgatttccaagcgcgcg cagagtgagccggagctgaagct Amylase-Phy- cttcatccccagcatcattacacctcagcaatggt F cgcctggtggtccctcttcctctacggtctccagg tcgccgcccccgccctcgccgccacccccgccgac tggcgctcccagagtgagccggagctgaagct

EXAMPLE 3

Construction of Codon-Optimized Escherichia coli Phytase Integrated Plasmid

[0062] Plasmids pGla-Phy-PhyOPT, pGla-Gla-PhyOPT, and pGla-Amy-PhyOPT were constructed respectively. Various signal peptide sequences including the Escherichia coli phytase signal peptide, Aspergillus niger glycosylase signal peptide, and Aspergillus oryzae TAKA amylase signal peptide were respectively linked to the codon-optimized Escherichia coli phytase sequence PhyOPT (SEQ ID NO. 8), and then used to replace the Aspergillus niger glycosylase gene. The phytase sequence derived from Escherichia coli ATCC 8739 was codon optimized to have a sequence as shown in SEQ ID NO. 8, which was synthesized by Nanjing Kingsray Biotechnology Co., Ltd. Similarly, the Phy signal peptide was optimized to have a sequence as shown in SEQ ID NO. 9. The integrated plasmid was constructed as follows. The pHphtk plasmid was linearized by vector-F and vector-R primers. The genome of Aspergillus niger (available from China Center of Industrial Culture Collection under Accession No. CICC2462) eras used as a template, and the Gla-5'-F and Gla-5'-R and the Gla-3'-F and Gla-3'-R were respectively used to amplify the 5' and 3' flanking sequences of the glycosylase gene, where each fragment was 2000 bp long. Optimized Escherichia coli phytase sequence PhyOPT was amplified using Phy-PhyOPT-F and Phy-PhyOPT-R, in which optimized Phy signal peptide sequence was introduced on the primer. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Phy fragment were recombined by Gibson Assembly.RTM. Master Mix Kit (E2611 New England Biolabs) to obtain an integrated plasmid pGla-Phy-PhyOPT, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 5. Gla-PhyOPT-F and Phy-PhyOPT-R were used as primers, and the PhyOPT fragment was used as a template to obtain a Gla-PhyOPT fragment by PCR amplification. In this fragment, the Aspergillus niger glycosylase signal peptide sequence was introduced. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Gla-PhyOPT fragment were recombined by Gibson Assembly.RTM. Master Mix Kit to obtain an integrated plasmid pGla-Gla-Phy, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 6. Amylase-PhyOPT-F and Phy-PhyOPT-R were used as primers and the PhyOPT fragment was used as a template to obtain an Amylase-PhyOPT fragment by PCR amplification. In this fragment, the Aspergillus oryzae TAKA amylase signal peptide sequence was introduced. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Amylase-PhyOPT fragment were recombined by Gibson Assembly.RTM. Master Mix Kit to obtain an integrated plasmid pGla-Amylase-PhyOPT, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 7. The 2 kb 5'-terminal flanking DNA sequence of the glycosylase gene is shown in SEQ ID NO. 20, and the 2 kb 3'-terminal flanking DNA sequence is shown in SEQ ID NO. 21. The sequences of the Phy-PhyOPT, Gla-PhyOPT, and Amy-PhyOPT expression cassettes are respectively as shown in SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27.

[0063] Related primer sequences are listed below:

TABLE-US-00002 Primer name Sequence (5'.fwdarw.3') vector-F gtacagtgaccggtgactctactggcatg vector-R gatgcattcgcgaggtaccgagctc Gla-5'-F aattcgagctcggtacctcgcgaatgcatcctacca atgctctcgaggattgcctgaacattgacattcggc Gla-5'-R tgctgaggtgtaatgatgctggg Gla-3'-F acaatcaatccatttcgctatagttaaaggatg Gla-3'-R catgccagaaagagtcaccggtcactgtacatggcc aatgtggtagccgttatcag Phy-PhyOPT- cttcatccccagcatcattacacctcagcaatgtcc F gacatgaagtccggtaacatctccatgaaggccatc ctgatccccttcctgtccctgctgatccccctgacc ccccagtccgccttcgcccagtccgaacccgagctg aagc Phy-PhyOPT- cctttaactatagcgaaatggattgattgtttagag R ggagcaggcggggatgc Gla-PhyOPT- cacatccccagcatcattacacctcagcaatgtcga F ccgatctctactcgccctgagcggcctcgtctgcac agggaggcaaatgtgataccaagcgcgcgcagtccg agcccgagctcaagc Amylase-Phy- cttcatccccagcatcattacacctcagcaatggtc OPT-F gcctggtggtccctcttcctctacggtctccagatc gccgcccccgccctcgccgccacccccgccgactgg cgacccagtccgagcccgagctcaagc

EXAMPLE 4

Construction of Codon-Optimized Escherichia coli Phytase Mutant Integrated Plasmid

[0064] U.S. Pat. No. 7,432,098 describes the Escherichia coli phytase mutant NOV9X, which has better heat resistance and is more suitable for use in the area of feed. NOV9X has 9 amino acid mutations compared to the Escherichia coli phytase in the present invention. In order to verify whether NOV9X can be efficiently expressed under the guidance of Aspergillus oryzae TAKA amylase signal, 17 base mutations were introduced to PhyOPT to obtain the DNA sequence of NOV9X, as shown in SEQ ID NO. 14. NOV9X has 98.6% sequence identity to the codon optimized Escherichia coli phytase mature peptide DNA sequence (SEQ ID NO. 8). NOV9X is synthesized by Nanjing Kingsray Biotechnology Co., Ltd., and the mature peptide sequence encoded thereby is shown in SEQ ID NO. 15. 43 base mutations were further introduced in NOV9X to form NOV9XM, as shown in SEQ ID NO. 16, which has 95.9% sequence identity to the codon-optimized Escherichia coli phytase mature peptide coding DNA sequence (SEQ ID NO. 8). NOV9X is synthesized by Nanjing Kingsray Biotechnology Co., Ltd., and the mature peptide sequence encoded thereby is shown in SEQ ID NO. 17. Plasmids pGla-Amy-PhyM1 and pGla-Amy-PhyM2 were constructed to integrate Amy-NOV9X and Amy-NOV9XM into Aspergillus niger glycosylase locus, respectively. The integrated plasmid was constructed as follows. The pHphtk plasmid was linearized by vector-F and vector-R primers. The genome of Aspergillus niger (from China Center of Industrial Culture Collection under Accession No. CICC2462) was used as a template, and the Gla-5'-F and Gla-5'-R and the Gla-3'-F and Gla-3'-R were respectively used to amplify the 5' and 3' flanking sequences of the glycosylase gene, where each fragment was 2000 bp long. Amylase-PhyOPT-F and Phy-PhyOPT-R were used as primers and the NOV9X and NOV9XM fragments were respectively used as a template to obtain Amylase-PhyM1 and Amylase-PhyM2 fragments by PCR amplification. In the two fragments, the Aspergillus oryzae TAKA amylase signal peptide sequence was introduced. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Amylase-PhyM1 fragment were recombined by Gibson Assembly.RTM. Master Mix Kit to obtain an integrated plasmid pGla-Amylase-PhyM1, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 8. The linearized pHphtk vector, the 5' and 3' flanking fragments of the glycosylase gene, and the Amylase-PhyM2 fragment were recombined by Gibson Assembly.RTM. Master Mix Kit to obtain an integrated plasmid pGla-Amylase-PhyM2, the sequence of which was confirmed by sequencing. Mapping of the plasmid is shown in FIG. 9. The 2 kb 5'-terminal flanking DNA sequence of the glycosylase gene is shown in SEQ ID NO. 20, and the 2 kb 3'-terminal flanking DNA sequence is shown in SEQ ID NO. 21. The Amy-PhyM1 and Amy-PhyM2 expression cassettes are respectively as shown in SEQ ID NO. 28 and SEQ ID NO. 29.

[0065] Related primer sequences are listed below:

TABLE-US-00003 Primer name Sequence (5'.fwdarw.3') vector-F gtacagtgaccgatgactctttctggcatg vector-R gatgcattcgcgaggtaccgagctc Gla-5c-F aattcgagctcggtacctcgcgaatgcatcct accaatgctctcgaggattgcctgaacattga cattcggc Gla-5'-R tgctgaggtgtaatgatgctggg Gla-3'-F acaatcaatccatttcgctatagttaaaggat g Gla-Y-R catgccagaaagagtcaccggtcactgtacat ggccaatgtggtagccgttatcagcttcatcc ccagcatcattacacctcagcaatggtcgcct ggtggtccctcttcctctacg Amylase-PhyOPT- gtctccaggtcgccgcccccgccctcgccgcc F acccccgccgactggcgctcccagtccgagcc cgagctcaagc Phy-PhyOPT-R cctttaactatagcgaaatggattgattgttt acagggagcaggcggggatgc

EXAMPLE 5

Integration of Each Expression Cassette into Aspergillus niger

[0066] The starting strain in this Example was AND4L, which was obtained by knocking out the glycosylase gene, the fungal amylase gene and the acid amylase gene from the CICC2462 strain. The Aspergillus niger gene knockout/knockin method could be implemented by referring to the technical method disclosed in the examples in CN 103937766A or CN 104962594A. The integration of PhyPhyOPT and PhyM into the glycosylasee locus in this example was achieved in the same manner as that in the example of CN 104962594A, i.e., by the method described by Delmas et al. (Appl Environ Microbiol. 2014, 80(11): 3484-7). Specifically, a circular DNA vector is used, comprising gla 5' and 3' flanking sequences, a selectable marker, a reverse selectable marker (or a negative selectable marker), and an Escherichia coli replication sequence, i.e. the plasmid as described in Examples 1 to 4. The circular vector was transferred into Aspergillus niger, and the recombinant strain was obtained by forward selection, and the knockout/knock-in strain was obtained by the reverse selectable marker.

[0067] Protoplast transformation was used to introduce pGla-Phy-Phy, pGla-Gla-Phy, pGla-Amy-Phy, pGla-Phy-PhyOPT, pGla-Gla-PhyOPT, pGla-Amy-PhyOPT, pGla-Amy-PhyM1 and pGla-Amy-PhyM2 separately. The specific steps were as follows.

[0068] Preparation of protoplasts: Aspergillus niger mycelium was cultured in a TZ liquid medium with rich nutrients (containing 0.8% of beef extract powder; 0.2% of yeast extract; 0.5% of peptone; 0.2% of NaCl; and 3% of sucrose; pH 5.8). The mycelium was filtered from the liquid culture by mira-cloth (Calbiochem) and washed with 0.7 M NaCl (pH 5.8). The mycelium was drained and transferred to an enzymatic hydrolyzing buffer (pH 5.8) containing 1% of cellulase (Sigma), 1% of helicase (Sigma) and 0.2% of lywallzyme (Sigma), and enzymatically hydrolyzed at 30.degree. C. and 65 rpm for 3 hrs. Then, the enzymatic hydrolyzing buffer containing the protoplast was placed on ice and filtered through four layers of lens paper. The obtained filtrate was mildly centrifuged at 3000 rpm for 10 minutes at 4.degree. C., and the supernatant was discarded. The protoplast attached to the tube wall was washed once with an STC buffer (containing 1 M D-Sorbitol, 50 mM CaCl.sub.2, 10 mM Tris, pH 7.5), and finally resuspended in an appropriate amount of the STC buffer.

[0069] 10 .mu.l (concentration: 100 ng/.mu.l) of the circular plasmids pGla-Phy-Phy, pGla-Gla-Phy, pGla-Amy-Phy, pGla-Phy-PhyOPT, pGla-Gla-PhyOPT, pGla-Amy-PhyOPT, pGla-Amy-PhyM 1 and pGla-Amy-PhyM2 were respectively added to 100 .mu.l of the protoplast suspension, mixed until uniform, and then stood for 25 min at room temperature. Then a total of 900 .mu.l of a PEG solution was added in 3 times, mixed until uniform and allowed to stand for 25 min at room temperature. The solution was centrifuged at room temperature for 10 min at 3000 rpm. The supernatant was discarded and the protoplast attached to the wall of the tube was resuspended in 1 ml of the STC buffer. The suspension was mixed with a TB3 medium (containing 0.3% of yeast extract, 0.3% of acidically hydrolyzed casein, 20% of sucrose, and 0.7% of agar) previously cooled to about 45.degree. C. and plated. After solidification, the plate was placed and cultured in an incubator at 34.degree. C. After 24 hrs, a layer of TB3 solid medium (containing 1% of agar, the remaining components being the same as above) containing 300 ng/.mu.l of hygromycin was further plated on the plate, and the plate was further incubated in an incubator at 34.degree. C. for 4-5 days. The transformants that grew out of the upper medium were the integrated transformants. Several integrated transformants were randomly picked and passaged respectively on TB3 solid medium containing 300 ng/.mu.l hygromycin. After incubation at a constant temperature of 34.degree. C. for 3 days, the mycelium was collected, frozen in liquid nitrogen, and then ground. Subsequently, the genomic DNA of the integrated transformant was extracted with a fungal genome extraction kit (Hangzhou Bori Technology Co., Ltd.). Finally, the genomic DNA of the integrated transformant was identified by PCR, in which the primers for identification were Pep-5test-F and Pep-5test-R, and Pep-3test-F and Pep-3test-R. The PCR product was sequenced and confirmed to be integrated into glycosylase locus.

[0070] Related primer sequences are listed below:

TABLE-US-00004 Primer name Sequence (5'.fwdarw.3') Phy-5test-F aatcgtgtccgcagatgtacttcac Phy-5test-R ataatcatccactgcacctcagagc Phy-3test-F tttcccagtcacgacgttgtaaaac Phy-3test-R aactcgaacagtgtaggtgcaatgtc

[0071] A suitable amount of the ground mycelium of the confirmed positive transformant was picked up into a centrifuge tube containing 1 ml of sterile water, and vortexed to form a mycelium suspension. 100 .mu.l was taken and coated onto a TB3 solid plate containing 10 .mu.M 5-F2dU (5-fluoro-2-deoxyuridine, manufacturer: Sigma), and incubated at a constant temperature of 34.degree. C. for 4-5 days. Knockout transformants were grown. The transformant should be unable to grow on 300 ng/.mu.l hygromycin-containing plates after two generations on 10 .mu.M 5-F2dU plates (to prevent impure transformants). Then the genomic DNA of the knockout transformant was identified by PCR, in which the primer sequences and the genome extraction method were the same as above. PCR identification using Pep-5test-F and Pep-3test-R showed that the positive transformant product is 5.5 kb and the negative transformant is 6.3 kb. The positive transformants were confirmed by sequencing the PCR products to obtain strains AND4L-Phy-Phy, AND4L-Gla-Phy, AND4L-Amy-Phy, AND4L-Phy-PhyOPT, AND4L-Gla-PhyOPT, AND4L-Amy-PhyOPT, AND4L-Amy-PhyM1 and AND4L-Amy-PhyM2.

EXAMPLE 6

Shake Flask Fermentation of Strains

[0072] The strains AND4L-Phy-Phy, AND4L-Gla-Phy, AND4L-Amy-Phy, AND4L-Phy-PhyOPT, AND4L-Gla-PhyOPT, AND4L-Amy-PhyOPT, AND4L-Amy-PhyM1 and AND4L-Amy-PhyM2 obtained in Example 5 were inoculated into a shake flask containing 50 ml of YPG medium (containing 2 g/L yeast extract, 2 g/L peptone, and 10% glucose) respectively, and cultured at 34.degree. C. and 220 rpm for six days. The supernatant was subjected to denaturing, polyacrylamide gel electrophoresis (SDS-PAGE). For the expression of each strain, see Table 1 for details.

TABLE-US-00005 TABLE 1 Expression of strains Whether the sequence is Strain Signal peptide optimized Expression AND4L-Phy- Escherichia coli Not optimized No expression Phy phytase AND4L-Gla- Aspergillus niger Not optimized No expression Phy glycosylase AND4L-Amy- Aspergillus oryzae Not optimized Low Phy TAKA amylase expression AND4L-Phy- Escherichia coli Optimized No expression PhyOPT phytase AND4L-Gla- Aspergillus niger Optimized No expression PhyOPT glycosylase AND4L-Amy- Aspergillus oryzae Optimized High PhyOPT TAKA amylase expression AND4L-Amy- Aspergillus oryzae Optimized High PhyM1 TAKA amylase expression AND4L-Amy- Aspergillus oryzae Optimized High PhyM2 TAKA amylase expression

[0073] As can be seen from Table 1, after the DNA encoding Escherichia coli phytase or a mutant thereof is codon optimized, the expression level in the supernatant is good under the guidance of Aspergillus oryzae TAKA amylase signal peptide, and no protein expression occurs in the presence of other signal peptide sequences. For the non-optimized sequence, the expression level is also very low under the guidance of Aspergillus oryzae TAKA amylase signal peptide, which proves that the optimization of the DNA sequence is also critical for the expression. Good expression can also be achieved after 17 and 50 mutations were introduced into the optimized sequence, respectively.

Sequence CWU 1

1

2911329DNAArtificial SequenceDNA sequence encoding Escherichia coli phytase 1atgtcagata tgaaaagcgg aaacatatcg atgaaagcga tcttaatccc atttttatct 60cttctgattc cgttaacccc gcaatctgca ttcgctcaga gtgagccgga gctgaagctg 120gaaagtgtgg tgattgtcag tcgtcatggt gtgcgtgctc caaccaaggc cacgcaactg 180atgcaggatg tcaccccaga cgcatggcca acctggccgg taaaactggg ttggctgaca 240ccgcgcggtg gtgagctaat cgcctatctc ggacattacc aacgccagcg tctggtagcc 300gacggattgc tggcgaaaaa gggctgcccg cagtctggtc aggtcgcgat tattgctgat 360gtcgacgagc gtacccgtaa aacaggcgaa gccttcgccg ccgggctggc acctgactgt 420gcaataaccg tacataccca ggcagatacg tccagtcccg atccgttatt taatcctcta 480aaaactggcg tttgccaact ggataactcg aacgtgactg acgcgatcct cagcagggca 540ggagggtcaa ttgctgactt taccgggcat cggcaaacgg cgtttcgcga actggaacgg 600gtgcttaatt ttccgcaatc aaacttgtgc cttaaacgtg agaaacagga cgaaagctgt 660tcattaacgc aggcattacc atcggaactc aaggtgagcg ccgacaatgt ctcattaacc 720ggtgcggtaa gcctcgcatc aatgctgacg gagatatttc tcctgcaaca agcacaggga 780atgccggagc cggggtgggg aaggatcacc gattcacacc agtggaacac cttgctaagt 840ttgcataacg cgcaatttta tttgctacaa cgcacgccag aggttgcccg cagccgcgcc 900accccgttat tagatttgat caagacagcg ttgacgcccc atccaccgca aaaacaggcg 960tatggtgtga cattacccac ttcagtgctg tttatcgccg gacacgatac taatctggca 1020aatctcggcg gcgcactgga gctcaactgg acgcttcccg gtcagccgga taacacgccg 1080ccaggtggtg aactggtgtt tgaacgctgg cgtcggctaa gcgataacag ccagtggatt 1140caggtttcgc tggtcttcca gactttacag cagatgcgtg ataaaacgcc gctgtcatta 1200aatacgccgc ccggagaggt gaaactgacc ctggcaggat gtgaagagcg aaatgcgcag 1260ggcatgtgtt cgttggcagg ttttacgcaa atcgtgaatg aagcacgcat accggcgtgc 1320agtttgtaa 13292442PRTArtificial SequenceAmino Acid sequence of Escherichia coli phytase 2Met Ser Asp Met Lys Ser Gly Asn Ile Ser Met Lys Ala Ile Leu Ile1 5 10 15Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr Pro Gln Ser Ala Phe Ala 20 25 30Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg 35 40 45His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val 50 55 60Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr65 70 75 80Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln 85 90 95Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln Ser 100 105 110Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr 115 120 125Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val 130 135 140His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu145 150 155 160Lys Thr Gly Val Cys Gln Leu Asp Asn Ser Asn Val Thr Asp Ala Ile 165 170 175Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln 180 185 190Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn 195 200 205Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln 210 215 220Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr225 230 235 240Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln 245 250 255Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser 260 265 270His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu 275 280 285Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu 290 295 300Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala305 310 315 320Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp 325 330 335Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu 340 345 350Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu 355 360 365Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu 370 375 380Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu385 390 395 400Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu 405 410 415Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val 420 425 430Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu 435 44031233DNAArtificial SequenceDNA sequence encoding Escherichia coli phytase mature peptide 3cagagtgagc cggagctgaa gctggaaagt gtggtgattg tcagtcgtca tggtgtgcgt 60gctccaacca aggccacgca actgatgcag gatgtcaccc cagacgcatg gccaacctgg 120ccggtaaaac tgggttggct gacaccgcgc ggtggtgagc taatcgccta tctcggacat 180taccaacgcc agcgtctggt agccgacgga ttgctggcga aaaagggctg cccgcagtct 240ggtcaggtcg cgattattgc tgatgtcgac gagcgtaccc gtaaaacagg cgaagccttc 300gccgccgggc tggcacctga ctgtgcaata accgtacata cccaggcaga tacgtccagt 360cccgatccgt tatttaatcc tctaaaaact ggcgtttgcc aactggataa ctcgaacgtg 420actgacgcga tcctcagcag ggcaggaggg tcaattgctg actttaccgg gcatcggcaa 480acggcgtttc gcgaactgga acgggtgctt aattttccgc aatcaaactt gtgccttaaa 540cgtgagaaac aggacgaaag ctgttcatta acgcaggcat taccatcgga actcaaggtg 600agcgccgaca atgtctcatt aaccggtgcg gtaagcctcg catcaatgct gacggagata 660tttctcctgc aacaagcaca gggaatgccg gagccggggt ggggaaggat caccgattca 720caccagtgga acaccttgct aagtttgcat aacgcgcaat tttatttgct acaacgcacg 780ccagaggttg cccgcagccg cgccaccccg ttattagatt tgatcaagac agcgttgacg 840ccccatccac cgcaaaaaca ggcgtatggt gtgacattac ccacttcagt gctgtttatc 900gccggacacg atactaatct ggcaaatctc ggcggcgcac tggagctcaa ctggacgctt 960cccggtcagc cggataacac gccgccaggt ggtgaactgg tgtttgaacg ctggcgtcgg 1020ctaagcgata acagccagtg gattcaggtt tcgctggtct tccagacttt acagcagatg 1080cgtgataaaa cgccgctgtc attaaatacg ccgcccggag aggtgaaact gaccctggca 1140ggatgtgaag agcgaaatgc gcagggcatg tgttcgttgg caggttttac gcaaatcgtg 1200aatgaagcac gcataccggc gtgcagtttg taa 12334410PRTArtificial SequenceMature peptide sequence of Escherichia coli phytase 4Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg1 5 10 15His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val 20 25 30Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr 35 40 45Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln 50 55 60Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln Ser65 70 75 80Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr 85 90 95Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val 100 105 110His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu 115 120 125Lys Thr Gly Val Cys Gln Leu Asp Asn Ser Asn Val Thr Asp Ala Ile 130 135 140Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln145 150 155 160Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn 165 170 175Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln 180 185 190Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr 195 200 205Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln 210 215 220Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser225 230 235 240His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu 245 250 255Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu 260 265 270Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala 275 280 285Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp 290 295 300Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu305 310 315 320Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu 325 330 335Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu 340 345 350Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu 355 360 365Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu 370 375 380Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val385 390 395 400Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu 405 410596DNAArtificial SequenceDNA sequence encoding Escherichia coli phytase signal peptide 5atgtcagata tgaaaagcgg aaacatatcg atgaaagcga tcttaatccc atttttatct 60cttctgattc cgttaacccc gcaatctgca ttcgct 96632PRTArtificial SequenceEscherichia coli phytase signal peptide sequence 6Met Ser Asp Met Lys Ser Gly Asn Ile Ser Met Lys Ala Ile Leu Ile1 5 10 15Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr Pro Gln Ser Ala Phe Ala 20 25 3071329DNAArtificial SequenceOptimized DNA sequence encoding Escherichia coli phytase 7atgtccgaca tgaagtccgg taacatctcc atgaaggcca tcctgatccc cttcctgtcc 60ctgctgatcc ccctgacccc ccagtccgcc ttcgcccagt ccgagcccga gctcaagctc 120gagtccgtcg tcatcgtctc ccgccacggt gtccgcgccc ccaccaaggc cacccagctc 180atgcaggacg tcacccccga cgcctggccc acctggcccg tcaagctcgg ttggctcacc 240ccccgcggtg gtgagctcat cgcctacctc ggtcactacc agcgccagcg cctcgtcgcc 300gacggtctcc tcgccaagaa gggttgcccc cagtccggtc aggtcgccat catcgccgac 360gtcgacgagc gcacccgcaa gaccggtgag gccttcgccg ccggtctcgc ccccgactgc 420gccatcaccg tccacaccca ggccgacacc tcctcccccg accccctctt caaccccctc 480aagaccggtg tctgccagct cgacaactcc aacgtcaccg acgccatcct ctcccgcgcc 540ggtggttcca tcgccgactt caccggtcac cgccagaccg ccttccgcga gctcgagcgc 600gtcctcaact tcccccagtc caacctctgc ctcaagcgcg agaagcagga cgagtcctgc 660tccctcaccc aggccctccc ctccgagctc aaggtctccg ccgacaacgt ctccctcacc 720ggtgccgtct ccctcgcctc catgctcacc gagatcttcc tcctccagca ggcccagggt 780atgcccgagc ccggttgggg tcgcatcacc gactcccacc agtggaacac cctcctctcc 840ctccacaacg cccagttcta cctcctccag cgcacccccg aggtcgcccg ctcccgcgcc 900acccccctcc tcgacctcat caagaccgcc ctcacccccc acccccccca gaagcaggcc 960tacggtgtca ccctccccac ctccgtcctc ttcatcgccg gtcacgacac caacctcgcc 1020aacctcggtg gtgccctcga gctcaactgg accctccccg gtcagcccga caacaccccc 1080cccggtggtg agctcgtctt cgagcgctgg cgccgcctct ccgacaactc ccagtggatc 1140caggtctccc tcgtcttcca gaccctccag cagatgcgcg acaagacccc cctctccctc 1200aacacccccc ccggtgaggt caagctcacc ctcgccggtt gcgaggagcg caacgcccag 1260ggtatgtgct ccctcgccgg tttcacccag atcgtcaacg aggcccgcat ccccgcctgc 1320tccctctaa 132981233DNAArtificial SequenceOptimized DNA sequence encoding Escherichia coli phytase mature peptide 8cagtccgagc ccgagctcaa gctcgagtcc gtcgtcatcg tctcccgcca cggtgtccgc 60gcccccacca aggccaccca gctcatgcag gacgtcaccc ccgacgcctg gcccacctgg 120cccgtcaagc tcggttggct caccccccgc ggtggtgagc tcatcgccta cctcggtcac 180taccagcgcc agcgcctcgt cgccgacggt ctcctcgcca agaagggttg cccccagtcc 240ggtcaggtcg ccatcatcgc cgacgtcgac gagcgcaccc gcaagaccgg tgaggccttc 300gccgccggtc tcgcccccga ctgcgccatc accgtccaca cccaggccga cacctcctcc 360cccgaccccc tcttcaaccc cctcaagacc ggtgtctgcc agctcgacaa ctccaacgtc 420accgacgcca tcctctcccg cgccggtggt tccatcgccg acttcaccgg tcaccgccag 480accgccttcc gcgagctcga gcgcgtcctc aacttccccc agtccaacct ctgcctcaag 540cgcgagaagc aggacgagtc ctgctccctc acccaggccc tcccctccga gctcaaggtc 600tccgccgaca acgtctccct caccggtgcc gtctccctcg cctccatgct caccgagatc 660ttcctcctcc agcaggccca gggtatgccc gagcccggtt ggggtcgcat caccgactcc 720caccagtgga acaccctcct ctccctccac aacgcccagt tctacctcct ccagcgcacc 780cccgaggtcg cccgctcccg cgccaccccc ctcctcgacc tcatcaagac cgccctcacc 840ccccaccccc cccagaagca ggcctacggt gtcaccctcc ccacctccgt cctcttcatc 900gccggtcacg acaccaacct cgccaacctc ggtggtgccc tcgagctcaa ctggaccctc 960cccggtcagc ccgacaacac cccccccggt ggtgagctcg tcttcgagcg ctggcgccgc 1020ctctccgaca actcccagtg gatccaggtc tccctcgtct tccagaccct ccagcagatg 1080cgcgacaaga cccccctctc cctcaacacc ccccccggtg aggtcaagct caccctcgcc 1140ggttgcgagg agcgcaacgc ccagggtatg tgctccctcg ccggtttcac ccagatcgtc 1200aacgaggccc gcatccccgc ctgctccctc taa 1233996DNAArtificial SequenceOptimized DNA sequence encoding Escherichia coli phytase signal peptide 9atgtccgaca tgaagtccgg taacatctcc atgaaggcca tcctgatccc cttcctgtcc 60ctgctgatcc ccctgacccc ccagtccgcc ttcgcc 961075DNAArtificial SequenceDNA sequence encoding Aspergillus niger glycosylase signal peptide 10atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt ggcaaatgtg 60atttccaagc gcgcg 751125PRTArtificial SequenceAmino acid sequence of Aspergillus niger glycosylase signal peptide 11Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly1 5 10 15Leu Ala Asn Val Ile Ser Lys Arg Ala 20 251284DNAArtificial SequenceDNA sequence encoding Aspergillus oryzae TAKA amylase signal peptide 12atggtcgcct ggtggtccct cttcctctac ggtctccagg tcgccgcccc cgccctcgcc 60gccacccccg ccgactggcg ctcc 841328PRTArtificial SequenceAmino acid sequence of Aspergillus oryzae TAKA amylase signal peptide 13Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala Ala1 5 10 15Pro Ala Leu Ala Ala Thr Pro Ala Asp Trp Arg Ser 20 25141233DNAArtificial SequenceOptimized DNA sequence encoding Escherichia coli phytase mutant NOV9X mature peptide 14cagtccgagc ccgagctcaa gctcgagtcc gtcgtcatcg tctcccgcca cggtgtccgc 60gcccccacca aggccaccca gctcatgcag gacgtcaccc ccgacgcctg gcccacctgg 120cccgtcaagc tcggtgagct caccccccgc ggtggtgagc tcatcgccta cctcggtcac 180tactggcgcc agcgcctcgt cgccgacggt ctcctcccca agtgcggttg cccccagtcc 240ggtcaggtcg ccatcatcgc cgacgtcgac gagcgcaccc gcaagaccgg tgaggccttc 300gccgccggtc tcgcccccga ctgcgccatc accgtccaca cccaggccga cacctcctcc 360cccgaccccc tcttcaaccc cctcaagacc ggtgtctgcc agctcgacaa cgccaacgtc 420accgacgcca tcctcgagcg cgccggtggt tccatcgccg acttcaccgg tcactaccag 480accgccttcc gcgagctcga gcgcgtcctc aacttccccc agtccaacct ctgcctcaag 540cgcgagaagc aggacgagtc ctgctccctc acccaggccc tcccctccga gctcaaggtc 600tccgccgact gcgtctccct caccggtgcc gtctccctcg cctccatgct caccgagatc 660ttcctcctcc agcaggccca gggtatgccc gagcccggtt ggggtcgcat caccgactcc 720caccagtgga acaccctcct ctccctccac aacgcccagt tcgacctcct ccagcgcacc 780cccgaggtcg cccgctcccg cgccaccccc ctcctcgacc tcatcaagac cgccctcacc 840ccccaccccc cccagaagca ggcctacggt gtcaccctcc ccacctccgt cctcttcatc 900gccggtcacg acaccaacct cgccaacctc ggtggtgccc tcgagctcaa ctggaccctc 960cccggtcagc ccgacaacac cccccccggt ggtgagctcg tcttcgagcg ctggcgccgc 1020ctctccgaca actcccagtg gatccaggtc tccctcgtct tccagaccct ccagcagatg 1080cgcgacaaga cccccctctc cctcaacacc ccccccggtg aggtcaagct caccctcgcc 1140ggttgcgagg agcgcaacgc ccagggtatg tgctccctcg ccggtttcac ccagatcgtc 1200aacgaggccc gcatccccgc ctgctccctc taa 123315410PRTArtificial SequenceOptimized Escherichia coli phytase mutant NOV9X mature peptide sequence 15Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg1 5 10 15His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val 20 25 30Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr 35 40 45Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln 50 55 60Arg Leu Val Ala Asp Gly Leu Leu Pro Lys Cys Gly Cys Pro Gln Ser65 70 75 80Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr 85 90 95Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val 100 105 110His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu 115 120 125Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala Ile 130 135 140Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln145 150 155 160Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn 165 170 175Leu Cys Leu Lys

Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln 180 185 190Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Cys Val Ser Leu Thr 195 200 205Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln 210 215 220Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser225 230 235 240His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu 245 250 255Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu 260 265 270Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala 275 280 285Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp 290 295 300Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu305 310 315 320Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu 325 330 335Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu 340 345 350Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu 355 360 365Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu 370 375 380Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val385 390 395 400Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu 405 410161233DNAArtificial SequenceOptimized DNA sequence encoding Escherichia coli phytase mutant NOV9XM mature peptide 16cagtccgagc ccgagctcaa gctccagtcc gtcgtcatcg tctcccgcca cggtgtccgc 60gcccccaccc gcgccaccca gctcatgcag aacgtcaccc ccgacgcctg gcccacctgg 120aaccagaccc tcggtgagct caccccccgc ggtggtgagc tcatcgccta cctcggtcac 180tactggcgcc agcgcctcgt cgccgacggt ctcctcccca accagacctg cccccagtcc 240ggtcaggtcg ccatcatcgc cgacaccgac gagcgcaccc gcaagaccgg tgaggccttc 300gccgccggtc tcgcccccga ctgcgccatc accgtccaca cccaggccga cacctcctcc 360cccgaccccc tcttcaaccc cctccgcacc ggtgtctgcc agctcgacaa cgccaacgtc 420accgacgcca tcctcgagcg cgccggtggt tccatcgccg acttcaccgg tcactaccag 480accgccttcc gcgagctcga gcgcgtcctc aacttctccc agtccaacct ctgcctcaag 540cgcgagaagg aggacgagtc ctgctccctc acccaggccc tcccctccga gctcaaggtc 600tccgccgact gcgtctccct caccggtgcc gtctccctcg cctccatgct caccgagatc 660ttcctcctcc agcaggccca gggtatgccc gagcccggtt ggggtcgcat caccgactcc 720caccagtgga acaccctcct ctccctccac aacgcccagt tcgacctcct caaccgcacc 780cccgaggtcg cccgctcccg cgccaccccc ctcctcgacc tcatcaagac cgccctcacc 840cccaacggta cccagaagca ggccaagggt gtcaccctcc ccacctccgt cctcttcatc 900gccggtcacg acaccaacct cgccaacctc ggtggtgccc tcgagctcaa ctggaccctc 960cccggtcagc ccgacaacac cccccccggt ggtgagctcg tcttcgagcg ctggcgccgc 1020ctctccgaca actcccagtg gatcaacgtc tccctcgtct tccagaccct ccagcagatg 1080cgcgacaaga cccccctctc cctcaacacc ccccccggtg aggtcaagct caccctcgcc 1140ggttgcgagg agcgcaacgc ccagggtatg tgctccctcg ccggtttcac ccagatcgtc 1200aacgaggccc gcctccccgc ctgctccctc taa 123317410PRTArtificial SequenceOptimized Escherichia coli phytase mutant NOV9XM mature peptide sequence 17Gln Ser Glu Pro Glu Leu Lys Leu Gln Ser Val Val Ile Val Ser Arg1 5 10 15His Gly Val Arg Ala Pro Thr Arg Ala Thr Gln Leu Met Gln Asn Val 20 25 30Thr Pro Asp Ala Trp Pro Thr Trp Asn Gln Thr Leu Gly Glu Leu Thr 35 40 45Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln 50 55 60Arg Leu Val Ala Asp Gly Leu Leu Pro Asn Gln Thr Cys Pro Gln Ser65 70 75 80Gly Gln Val Ala Ile Ile Ala Asp Thr Asp Glu Arg Thr Arg Lys Thr 85 90 95Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val 100 105 110His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu 115 120 125Arg Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala Ile 130 135 140Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln145 150 155 160Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Ser Gln Ser Asn 165 170 175Leu Cys Leu Lys Arg Glu Lys Glu Asp Glu Ser Cys Ser Leu Thr Gln 180 185 190Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Cys Val Ser Leu Thr 195 200 205Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln 210 215 220Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser225 230 235 240His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu 245 250 255Leu Asn Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu 260 265 270Asp Leu Ile Lys Thr Ala Leu Thr Pro Asn Gly Thr Gln Lys Gln Ala 275 280 285Lys Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp 290 295 300Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu305 310 315 320Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu 325 330 335Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Asn Val Ser Leu 340 345 350Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu 355 360 365Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu 370 375 380Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val385 390 395 400Asn Glu Ala Arg Leu Pro Ala Cys Ser Leu 405 410182515DNAArtificial Sequencehph gene expression cassette 18gtacagtgac cggtgactct ttctggcatg cggagagacg gacggacgca gagagaaggg 60ctgagtaata agccactggc cagacagctc tggcggctct gaggtgcagt ggatgattat 120taatccggga ccggccgccc ctccgccccg aagtggaaag gctggtgtgc ccctcgttga 180ccaagaatct attgcatcat cggagaatat ggagcttcat cgaatcaccg gcagtaagcg 240aaggagaatg tgaagccagg ggtgtatagc cgtcggcgaa atagcatgcc attaacctag 300gtacagaagt ccaattgctt ccgatctggt aaaagattca cgagatagta ccttctccga 360agtaggtaga gcgagtaccc ggcgcgtaag ctccctaatt ggcccatccg gcatctgtag 420ggcgtccaaa tatcgtgcct ctcctgcttt gcccggtgta tgaaaccgga aaggccgctc 480aggagctggc cagcggcgca gaccgggaac acaagctggc agtcgaccca tccggtgctc 540tgcactcgac ctgctgaggt ccctcagtcc ctggtaggca gctttgcccc gtctgtccgc 600ccggtgtgtc ggcggggttg acaaggtcgt tgcgtcagtc caacatttgt tgccatattt 660tcctgctctc cccaccagct gctcttttct tttctctttc ttttcccatc ttcagtatat 720tcatcttccc atccaagaac ctttatttcc cctaagtaag tactttgcta catccatact 780ccatccttcc catcccttat tcctttgaac ctttcagttc gagctttccc acttcatcgc 840agcttgacta acagctaccc cgcttgagca gacatcacca tgaaaaagcc tgaactcacc 900gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 960ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 1020ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 1080gcatcggccg cgctcccgat tccggaagtg cttgacattg gggagttcag cgagagcctg 1140acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 1200ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt 1260agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 1320cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 1380gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 1440tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 1500aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 1560gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 1620tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 1680cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 1740tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 1800caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 1860agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgatt taatagctcc 1920atgtcaacaa gaataaaacg cgttttcggg tttacctctt ccagatacag ctcatctgca 1980atgcattaat gcattgactg caacctagta acgccttcag gctccggcga agagaagaat 2040agcttagcag agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa 2100gagacctacg agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa 2160ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac 2220cagccctggg ttcgcaaaga taattgcatg tttcttcctt gaactctcaa gcctacagga 2280cacacattca tcgtaggtat aaacctcgaa atcattccta ctaagatggt atacaatagt 2340aaccatggtt gcctagtgaa tgctccgtaa cacccaatac gccggccgaa acttttttac 2400aactctccta tgagtcgttt acccagaatg cacaggtaca cttgtttaga ggtaatcctt 2460ctttctagaa gtcctcgtgt actgtgtaag cgcccactcc acatctccac tcgag 2515191979DNAArtificial SequenceHSV-tk expression cassette 19ggatcccggg tctacgccag gaccgagcaa gcccagatga gaaccgacgc agatttcctt 60ggcacctgtt gcttcagctg aatcctggca atacgagata cctgctttga atattttgaa 120tagctcgccc gctggagagc atcctgaatg caagtaacaa ccgtagaggc tgacacggca 180ggtgttgcta gggagcgtcg tgttctacaa ggccagacgt cttcgcggtt gatatatatg 240tatgtttgac tgcaggctgc tcagcgacga cagtcaagtt cgccctcgct gcttgtgcaa 300taatcgcagt ggggaagcca caccgtgact cccatctttc agtaaagctc tgttggtgtt 360tatcagcaat acacgtaatt taaactcgtt agcatggggc tgatagctta attaccgttt 420accagtgccg cggttctgca gctttccttg gcccgtaaaa ttcggcgaag ccagccaatc 480accagctagg caccagctaa accctataat tagtctctta tcaacaccat ccgctccccc 540gggatcaatg aggagaatga gggggatgcg gggctaaaga agcctacata accctcatgc 600caactcccag tttacactcg tcgagccaac atcctgacta taagctaaca cagaatggct 660tcgtacccct gccatcaaca cgcgtctgcg ttcgaccagg ctgcgcgttc tcgcggccat 720aacaaccgac gtacggcgtt gcgccctcgc cggcaacaaa aagccacgga agtccgcctg 780gagcagaaaa tgcccacgct actgcgggtt tatatagacg gtccccacgg gatggggaaa 840accaccacca cgcaactgct ggtggccctg ggttcgcgcg acgatatcgt ctacgtaccc 900gagccgatga cttactggcg ggtgttgggg gcttccgaga caatcgcgaa catctacacc 960acacaacacc gcctcgacca gggtgagata tcggccgggg acgcggcggt ggtaatgaca 1020agcgcccaga taacaatggg catgccttat gccgtgaccg acgccgttct ggctcctcat 1080atcggggggg aggctgggag ctcacatgcc ccgcccccgg ccctcaccct catcttcgac 1140cgccatccca tcgccgccct cctgtgctac ccggccgcgc gataccttat gggcagcatg 1200accccccagg ccgtgctggc gttcgtggcc ctcatcccgc cgaccttgcc cggcacaaac 1260atcgtgttgg gggcccttcc ggaggacaga cacatcgacc gcctggccaa acgccagcgc 1320cccggcgagc ggcttgacct ggctatgctg gccgcgattc gccgcgttta tgggctgctt 1380gccaatacgg tgcggtatct gcagggcggc gggtcgtggc gggaggattg gggacagctt 1440tcgggggcgg ccgtgccgcc ccagggtgcc gagccccaga gcaacgcggg cccacgaccc 1500catatcgggg acacgttatt taccctgttt cgggcccccg agttgctggc ccccaacggc 1560gacctgtata acgtgtttgc ctgggctttg gacgtcttgg ccaaacgcct ccgtcccatg 1620catgtcttta tcctggatta cgaccaatcg cccgccggct gccgggacgc cctgctgcaa 1680cttacctccg ggatggtcca gacccacgtc accaccccag gctccatacc gacgatctgc 1740gacctggcgc gcacgtttgc ccgggagatg ggggaggcta actgactaat aagtgtcaga 1800tagcaatttg cacaagaaat caataccagc aactgtaaat aagcgctgaa gtgaccatgc 1860catgctacga aagagcagaa aaaaacctgc cgtagaaccg aagagatatg acacgcttcc 1920atctctcaaa ggaagaatcc cttcagggtt gcgtttccag gcggccgcaa attacatgt 1979202000DNAArtificial Sequence5'-terminal flanking sequence of glycosylase gene 20ctaccaatgc tctcgaggat tgcctgaaca ttgacattcg gcgtccggcc gggaccaccg 60cggactcgaa gctgcctgtg ctggtctgga tctttggcgg aggctttgaa cttggttcaa 120aggcgatgta tgatggtaca acgatggtat catcgtcgat agacaagaac atgcctatcg 180tgtttgtagc aatgaattat cgcgtgggag gtttcgggtt cttgcccgga aaggagatcc 240tggaggacgg gtccgcgaac ctagggctcc tggaccaacg ccttgccctg cagtgggttg 300ccgacaacat cgaggccttt ggtggagacc cggacaaggt gacgatttgg ggagaatcag 360caggagccat ttccgttttt gatcagatga tcttgtacga cggaaacatc acttacaagg 420ataagccctt gttccggggg gccatcatgg actccggtag tgttgttccc gcagaccccg 480tcgatggggt caagggacag caagtatatg atgcggtagt ggaatctgca ggctgttcct 540cttctaacga caccctagct tgtctgcgtg aactagacta caccgacttc ctcaatgcgg 600caaactccgt gccaggcatt ttaagctacc attctgtggc gttatcatat gtgcctcgac 660cggacgggac ggcgttgtcg gcatcaccgg acgttttggg caaagcaggg aaatatgctc 720gggtcccgtt catcgtgggc gaccaagagg atgaggggac cttattcgcc ttgtttcagt 780ccaacattac gacgatcgac gaggtggtcg actacctggc ctcatacttc ttctatgacg 840ctagccgaga gcagcttgaa gaactagtgg ccctgtaccc agacaccacc acgtacgggt 900ctccgttcag gacaggcgcg gccaacaact ggtatccgca atttaagcga ttggccgcca 960ttctcggcga cttggtcttc accattaccc ggcgggcatt cctctcgtat gcagaggaaa 1020tctcccctga tcttccgaac tggtcgtacc tggcgaccta tgactatggc accccagttc 1080tggggacctt ccacggaagt gacctgctgc aggtgttcta tgggatcaag ccaaactatg 1140cagctagttc tagccacacg tactatctga gctttgtgta tacgctggat ccgaactcca 1200accgggggga gtacattgag tggccgcagt ggaaggaatc gcggcagttg atgaatttcg 1260gagcgaacga cgccagtctc cttacggatg atttccgcaa cgggacatat gagttcatcc 1320tgcagaatac cgcggcgttc cacatctgat gccattggcg gaggggtccg gacggtcagg 1380aacttagcct tatgagatga atgatggacg tgtctggcct cggaaaagga tatatgggga 1440tcatgatagt actagccata ttaatgaagg gcatatacca cgcgttggac ctgcgttata 1500gcttcccgtt agttatagta ccatcgttat accagccaat caagtcacca cgcacgaccg 1560gggacggcga atccccggga attgaaagaa attgcatccc aggccagtga ggccagcgat 1620tggccacctc tccaaggcac agggccattc tgcagcgctg gtggattcat cgcaatttcc 1680cccggcccgg cccgacaccg ctataggctg gttctcccac accatcggag attcgtcgcc 1740taatgtctcg tccgttcaca agctgaagag cttgaagtgg cgagatgtct ctgcaggaat 1800tcaagctaga tgctaagcga tattgcatgg caatatgtgt tgatgcatgt gcttcttcct 1860tcagcttccc ctcgtgcaga tgaggtttgg ctataaattg aagtggttgg tcggggttcc 1920gtgaggggct gaagtgcttc ctccctttta gacgcaactg agagcctgag cttcatcccc 1980agcatcatta cacctcagca 2000212000DNAArtificial Sequence3'-terminal flanking sequence of glycosylase gene 21acaatcaatc catttcgcta tagttaaagg atggggatga gggcaattgg ttatatgatc 60atgtatgtag tgggtgtgca taatagtagt gaaatggaag ccaagtcatg tgattgtaat 120cgaccgacgg aattgaggat atccggaaat acagacaccg tgaaagccat ggtctttcct 180tcgtgtagaa gaccagacag acagtccctg atttaccctt gcacaaagca ctagaaaatt 240agcattccat ccttctctgc ttgctctgct gatatcactg tcattcaatg catagccatg 300agctcatctt agatccaagc acgtaattcc atagccgagg tccacagtgg agcagcaaca 360ttccccatca ttgctttccc caggggcctc ccaacgacta aatcaagagt atatctctac 420cgtccaatag atcgtcttcg cttcaaaatc tttgacaatt ccaagagggt ccccatccat 480caaacccagt tcaataatag ccgagatgca tggtggagtc aattaggcag tattgctgga 540atgtcggggc cagttggccc ggtggtcatt ggccgcctgt gatgccatct gccactaaat 600ccgatcattg atccaccgcc cacgaggcgc gtctttgctt tttgcgcggc gtccaggttc 660aactctctct gcagctccag tccaacgctg actgactagt ttacctactg gtctgatcgg 720ctccatcaga gctatggcgt tatcccgtgc cgttgctgcg caatcgctat cttgatcgca 780accttgaact cactcttgtt ttaatagtga tcttggtgac ggagtgtcgg tgagtgacaa 840ccaacatcgt gcaagggaga ttgatacgga attgtcgctc ccatcatgat gttcttgccg 900gctttgttgg ccctattcgt gggatgcgat gccctcgctg tgcagcagca ggtactgctg 960gatgaggagc catcggtctc tgcacgcaaa cccaacttcc tcttcattct cacggatgat 1020caggatctcc ggatgaattc tccggcgtat atgccgtata cgcaggcgag aatcaaggaa 1080aagggtaccg agttcttgaa ccatttcgtc actaccgcgc tttgctgtcc gtcgcgcgtg 1140agtctttgga cgggaagaca ggctcataat actaatgtga cggatgtgaa cccgccttat 1200ggtatggaca ctgcttcgat cggtcttgat tcttcagcgt ggttacaatt gctaatgcgg 1260cataggcgga taccccaaat tcgtcgctca aggcttcaac gaaaacttcc tccccgtttg 1320gctgcagtcc gccggttaca atacctacta cacggggaag ctgttcaact cgcacagtgt 1380cgctacctat aacgcgccct ttgtgaacgg tttcaatggc tccgacttcc tcctcgaccc 1440ccacacatat tcctactgga atgcgacata ccagcgaaac catgagcctc cgcggagtta 1500cgagggacaa tatactacgg atgtgatgaa ggagaaggca tcgggattgt tggcagatgc 1560gctggacagt gacgcgccat tcttcctgac ggtcgcgccg atcgcaccgc acacgaacat 1620cgatgtggag gggctgagcg gtgcgggtgg accgaagatg acagagccgc tgcctgcacc 1680gagacatgcg catttgtttg ctgatgcaaa ggtgccgcgg acgcctaatt tcaatccgga 1740caaggtgtgt gatatcctga cacagtggtg gggacgggca ctgacaagag taggattctg 1800gtgcggggtg gatccaaacc atggaactac agaaccagac cgtcatcgac tacgaagacc 1860atctttatcg ccagcgtctg cgcactttgc aagccgtcga tgagatggtg gatgcgctga 1920tcacgcagct ggaagaaagt gggcagatcg acaataccta catcatttac agtgctgata 1980acggctacca cattggccat 2000222480DNAArtificial SequencePhy-Phy expression cassette 22tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatgtcagat 660atgaaaagcg gaaacatatc gatgaaagcg atcttaatcc catttttatc tcttctgatt 720ccgttaaccc cgcaatctgc attcgctcag agtgagccgg agctgaagct ggaaagtgtg 780gtgattgtca gtcgtcatgg tgtgcgtgct

ccaaccaagg ccacgcaact gatgcaggat 840gtcaccccag acgcatggcc aacctggccg gtaaaactgg gttggctgac accgcgcggt 900ggtgagctaa tcgcctatct cggacattac caacgccagc gtctggtagc cgacggattg 960ctggcgaaaa agggctgccc gcagtctggt caggtcgcga ttattgctga tgtcgacgag 1020cgtacccgta aaacaggcga agccttcgcc gccgggctgg cacctgactg tgcaataacc 1080gtacataccc aggcagatac gtccagtccc gatccgttat ttaatcctct aaaaactggc 1140gtttgccaac tggataactc gaacgtgact gacgcgatcc tcagcagggc aggagggtca 1200attgctgact ttaccgggca tcggcaaacg gcgtttcgcg aactggaacg ggtgcttaat 1260tttccgcaat caaacttgtg ccttaaacgt gagaaacagg acgaaagctg ttcattaacg 1320caggcattac catcggaact caaggtgagc gccgacaatg tctcattaac cggtgcggta 1380agcctcgcat caatgctgac ggagatattt ctcctgcaac aagcacaggg aatgccggag 1440ccggggtggg gaaggatcac cgattcacac cagtggaaca ccttgctaag tttgcataac 1500gcgcaatttt atttgctaca acgcacgcca gaggttgccc gcagccgcgc caccccgtta 1560ttagatttga tcaagacagc gttgacgccc catccaccgc aaaaacaggc gtatggtgtg 1620acattaccca cttcagtgct gtttatcgcc ggacacgata ctaatctggc aaatctcggc 1680ggcgcactgg agctcaactg gacgcttccc ggtcagccgg ataacacgcc gccaggtggt 1740gaactggtgt ttgaacgctg gcgtcggcta agcgataaca gccagtggat tcaggtttcg 1800ctggtcttcc agactttaca gcagatgcgt gataaaacgc cgctgtcatt aaatacgccg 1860cccggagagg tgaaactgac cctggcagga tgtgaagagc gaaatgcgca gggcatgtgt 1920tcgttggcag gttttacgca aatcgtgaat gaagcacgca taccggcgtg cagtttgtaa 1980tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 2040gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 2100ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 2160taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 2220aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 2280ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 2340ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 2400gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 2460gcaatatgtg ttgatgcatg 2480232459DNAArtificial SequenceGla-Phy expression cassette 23tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatgtcgttc 660cgatctctac tcgccctgag cggcctcgtc tgcacagggt tggcaaatgt gatttccaag 720cgcgcgcaga gtgagccgga gctgaagctg gaaagtgtgg tgattgtcag tcgtcatggt 780gtgcgtgctc caaccaaggc cacgcaactg atgcaggatg tcaccccaga cgcatggcca 840acctggccgg taaaactggg ttggctgaca ccgcgcggtg gtgagctaat cgcctatctc 900ggacattacc aacgccagcg tctggtagcc gacggattgc tggcgaaaaa gggctgcccg 960cagtctggtc aggtcgcgat tattgctgat gtcgacgagc gtacccgtaa aacaggcgaa 1020gccttcgccg ccgggctggc acctgactgt gcaataaccg tacataccca ggcagatacg 1080tccagtcccg atccgttatt taatcctcta aaaactggcg tttgccaact ggataactcg 1140aacgtgactg acgcgatcct cagcagggca ggagggtcaa ttgctgactt taccgggcat 1200cggcaaacgg cgtttcgcga actggaacgg gtgcttaatt ttccgcaatc aaacttgtgc 1260cttaaacgtg agaaacagga cgaaagctgt tcattaacgc aggcattacc atcggaactc 1320aaggtgagcg ccgacaatgt ctcattaacc ggtgcggtaa gcctcgcatc aatgctgacg 1380gagatatttc tcctgcaaca agcacaggga atgccggagc cggggtgggg aaggatcacc 1440gattcacacc agtggaacac cttgctaagt ttgcataacg cgcaatttta tttgctacaa 1500cgcacgccag aggttgcccg cagccgcgcc accccgttat tagatttgat caagacagcg 1560ttgacgcccc atccaccgca aaaacaggcg tatggtgtga cattacccac ttcagtgctg 1620tttatcgccg gacacgatac taatctggca aatctcggcg gcgcactgga gctcaactgg 1680acgcttcccg gtcagccgga taacacgccg ccaggtggtg aactggtgtt tgaacgctgg 1740cgtcggctaa gcgataacag ccagtggatt caggtttcgc tggtcttcca gactttacag 1800cagatgcgtg ataaaacgcc gctgtcatta aatacgccgc ccggagaggt gaaactgacc 1860ctggcaggat gtgaagagcg aaatgcgcag ggcatgtgtt cgttggcagg ttttacgcaa 1920atcgtgaatg aagcacgcat accggcgtgc agtttgtaat gccattggcg gaggggtccg 1980gacggtcagg aacttagcct tatgagatga atgatggacg tgtctggcct cggaaaagga 2040tatatgggga tcatgatagt actagccata ttaatgaagg gcatatacca cgcgttggac 2100ctgcgttata gcttcccgtt agttatagta ccatcgttat accagccaat caagtcacca 2160cgcacgaccg gggacggcga atccccggga attgaaagaa attgcatccc aggccagtga 2220ggccagcgat tggccacctc tccaaggcac agggccattc tgcagcgctg gtggattcat 2280cgcaatttcc cccggcccgg cccgacaccg ctataggctg gttctcccac accatcggag 2340attcgtcgcc taatgtctcg tccgttcaca agctgaagag cttgaagtgg cgagatgtct 2400ctgcaggaat tcaagctaga tgctaagcga tattgcatgg caatatgtgt tgatgcatg 2459242468DNAArtificial SequenceAmy-Phy expression cassette 24tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatggtcgcc 660tggtggtccc tcttcctcta cggtctccag gtcgccgccc ccgccctcgc cgccaccccc 720gccgactggc gctcccagag tgagccggag ctgaagctgg aaagtgtggt gattgtcagt 780cgtcatggtg tgcgtgctcc aaccaaggcc acgcaactga tgcaggatgt caccccagac 840gcatggccaa cctggccggt aaaactgggt tggctgacac cgcgcggtgg tgagctaatc 900gcctatctcg gacattacca acgccagcgt ctggtagccg acggattgct ggcgaaaaag 960ggctgcccgc agtctggtca ggtcgcgatt attgctgatg tcgacgagcg tacccgtaaa 1020acaggcgaag ccttcgccgc cgggctggca cctgactgtg caataaccgt acatacccag 1080gcagatacgt ccagtcccga tccgttattt aatcctctaa aaactggcgt ttgccaactg 1140gataactcga acgtgactga cgcgatcctc agcagggcag gagggtcaat tgctgacttt 1200accgggcatc ggcaaacggc gtttcgcgaa ctggaacggg tgcttaattt tccgcaatca 1260aacttgtgcc ttaaacgtga gaaacaggac gaaagctgtt cattaacgca ggcattacca 1320tcggaactca aggtgagcgc cgacaatgtc tcattaaccg gtgcggtaag cctcgcatca 1380atgctgacgg agatatttct cctgcaacaa gcacagggaa tgccggagcc ggggtgggga 1440aggatcaccg attcacacca gtggaacacc ttgctaagtt tgcataacgc gcaattttat 1500ttgctacaac gcacgccaga ggttgcccgc agccgcgcca ccccgttatt agatttgatc 1560aagacagcgt tgacgcccca tccaccgcaa aaacaggcgt atggtgtgac attacccact 1620tcagtgctgt ttatcgccgg acacgatact aatctggcaa atctcggcgg cgcactggag 1680ctcaactgga cgcttcccgg tcagccggat aacacgccgc caggtggtga actggtgttt 1740gaacgctggc gtcggctaag cgataacagc cagtggattc aggtttcgct ggtcttccag 1800actttacagc agatgcgtga taaaacgccg ctgtcattaa atacgccgcc cggagaggtg 1860aaactgaccc tggcaggatg tgaagagcga aatgcgcagg gcatgtgttc gttggcaggt 1920tttacgcaaa tcgtgaatga agcacgcata ccggcgtgca gtttgtaatg ccattggcgg 1980aggggtccgg acggtcagga acttagcctt atgagatgaa tgatggacgt gtctggcctc 2040ggaaaaggat atatggggat catgatagta ctagccatat taatgaaggg catataccac 2100gcgttggacc tgcgttatag cttcccgtta gttatagtac catcgttata ccagccaatc 2160aagtcaccac gcacgaccgg ggacggcgaa tccccgggaa ttgaaagaaa ttgcatccca 2220ggccagtgag gccagcgatt ggccacctct ccaaggcaca gggccattct gcagcgctgg 2280tggattcatc gcaatttccc ccggcccggc ccgacaccgc tataggctgg ttctcccaca 2340ccatcggaga ttcgtcgcct aatgtctcgt ccgttcacaa gctgaagagc ttgaagtggc 2400gagatgtctc tgcaggaatt caagctagat gctaagcgat attgcatggc aatatgtgtt 2460gatgcatg 2468252480DNAArtificial SequencePhy-PhyOpt expression cassette 25tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatgtccgac 660atgaagtccg gtaacatctc catgaaggcc atcctgatcc ccttcctgtc cctgctgatc 720cccctgaccc cccagtccgc cttcgcccag tccgagcccg agctcaagct cgagtccgtc 780gtcatcgtct cccgccacgg tgtccgcgcc cccaccaagg ccacccagct catgcaggac 840gtcacccccg acgcctggcc cacctggccc gtcaagctcg gttggctcac cccccgcggt 900ggtgagctca tcgcctacct cggtcactac cagcgccagc gcctcgtcgc cgacggtctc 960ctcgccaaga agggttgccc ccagtccggt caggtcgcca tcatcgccga cgtcgacgag 1020cgcacccgca agaccggtga ggccttcgcc gccggtctcg cccccgactg cgccatcacc 1080gtccacaccc aggccgacac ctcctccccc gaccccctct tcaaccccct caagaccggt 1140gtctgccagc tcgacaactc caacgtcacc gacgccatcc tctcccgcgc cggtggttcc 1200atcgccgact tcaccggtca ccgccagacc gccttccgcg agctcgagcg cgtcctcaac 1260ttcccccagt ccaacctctg cctcaagcgc gagaagcagg acgagtcctg ctccctcacc 1320caggccctcc cctccgagct caaggtctcc gccgacaacg tctccctcac cggtgccgtc 1380tccctcgcct ccatgctcac cgagatcttc ctcctccagc aggcccaggg tatgcccgag 1440cccggttggg gtcgcatcac cgactcccac cagtggaaca ccctcctctc cctccacaac 1500gcccagttct acctcctcca gcgcaccccc gaggtcgccc gctcccgcgc cacccccctc 1560ctcgacctca tcaagaccgc cctcaccccc cacccccccc agaagcaggc ctacggtgtc 1620accctcccca cctccgtcct cttcatcgcc ggtcacgaca ccaacctcgc caacctcggt 1680ggtgccctcg agctcaactg gaccctcccc ggtcagcccg acaacacccc ccccggtggt 1740gagctcgtct tcgagcgctg gcgccgcctc tccgacaact cccagtggat ccaggtctcc 1800ctcgtcttcc agaccctcca gcagatgcgc gacaagaccc ccctctccct caacaccccc 1860cccggtgagg tcaagctcac cctcgccggt tgcgaggagc gcaacgccca gggtatgtgc 1920tccctcgccg gtttcaccca gatcgtcaac gaggcccgca tccccgcctg ctccctctaa 1980tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 2040gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 2100ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 2160taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 2220aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 2280ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 2340ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 2400gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 2460gcaatatgtg ttgatgcatg 2480262459DNAArtificial SequenceGla-PhyOPT expression cassette 26tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatgtcgttc 660cgatctctac tcgccctgag cggcctcgtc tgcacagggt tggcaaatgt gatttccaag 720cgcgcgcagt ccgagcccga gctcaagctc gagtccgtcg tcatcgtctc ccgccacggt 780gtccgcgccc ccaccaaggc cacccagctc atgcaggacg tcacccccga cgcctggccc 840acctggcccg tcaagctcgg ttggctcacc ccccgcggtg gtgagctcat cgcctacctc 900ggtcactacc agcgccagcg cctcgtcgcc gacggtctcc tcgccaagaa gggttgcccc 960cagtccggtc aggtcgccat catcgccgac gtcgacgagc gcacccgcaa gaccggtgag 1020gccttcgccg ccggtctcgc ccccgactgc gccatcaccg tccacaccca ggccgacacc 1080tcctcccccg accccctctt caaccccctc aagaccggtg tctgccagct cgacaactcc 1140aacgtcaccg acgccatcct ctcccgcgcc ggtggttcca tcgccgactt caccggtcac 1200cgccagaccg ccttccgcga gctcgagcgc gtcctcaact tcccccagtc caacctctgc 1260ctcaagcgcg agaagcagga cgagtcctgc tccctcaccc aggccctccc ctccgagctc 1320aaggtctccg ccgacaacgt ctccctcacc ggtgccgtct ccctcgcctc catgctcacc 1380gagatcttcc tcctccagca ggcccagggt atgcccgagc ccggttgggg tcgcatcacc 1440gactcccacc agtggaacac cctcctctcc ctccacaacg cccagttcta cctcctccag 1500cgcacccccg aggtcgcccg ctcccgcgcc acccccctcc tcgacctcat caagaccgcc 1560ctcacccccc acccccccca gaagcaggcc tacggtgtca ccctccccac ctccgtcctc 1620ttcatcgccg gtcacgacac caacctcgcc aacctcggtg gtgccctcga gctcaactgg 1680accctccccg gtcagcccga caacaccccc cccggtggtg agctcgtctt cgagcgctgg 1740cgccgcctct ccgacaactc ccagtggatc caggtctccc tcgtcttcca gaccctccag 1800cagatgcgcg acaagacccc cctctccctc aacacccccc ccggtgaggt caagctcacc 1860ctcgccggtt gcgaggagcg caacgcccag ggtatgtgct ccctcgccgg tttcacccag 1920atcgtcaacg aggcccgcat ccccgcctgc tccctctaat gccattggcg gaggggtccg 1980gacggtcagg aacttagcct tatgagatga atgatggacg tgtctggcct cggaaaagga 2040tatatgggga tcatgatagt actagccata ttaatgaagg gcatatacca cgcgttggac 2100ctgcgttata gcttcccgtt agttatagta ccatcgttat accagccaat caagtcacca 2160cgcacgaccg gggacggcga atccccggga attgaaagaa attgcatccc aggccagtga 2220ggccagcgat tggccacctc tccaaggcac agggccattc tgcagcgctg gtggattcat 2280cgcaatttcc cccggcccgg cccgacaccg ctataggctg gttctcccac accatcggag 2340attcgtcgcc taatgtctcg tccgttcaca agctgaagag cttgaagtgg cgagatgtct 2400ctgcaggaat tcaagctaga tgctaagcga tattgcatgg caatatgtgt tgatgcatg 2459272468DNAArtificial SequenceAmy-PhyOPT expression cassette 27tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatggtcgcc 660tggtggtccc tcttcctcta cggtctccag gtcgccgccc ccgccctcgc cgccaccccc 720gccgactggc gctcccagtc cgagcccgag ctcaagctcg agtccgtcgt catcgtctcc 780cgccacggtg tccgcgcccc caccaaggcc acccagctca tgcaggacgt cacccccgac 840gcctggccca cctggcccgt caagctcggt tggctcaccc cccgcggtgg tgagctcatc 900gcctacctcg gtcactacca gcgccagcgc ctcgtcgccg acggtctcct cgccaagaag 960ggttgccccc agtccggtca ggtcgccatc atcgccgacg tcgacgagcg cacccgcaag 1020accggtgagg ccttcgccgc cggtctcgcc cccgactgcg ccatcaccgt ccacacccag 1080gccgacacct cctcccccga ccccctcttc aaccccctca agaccggtgt ctgccagctc 1140gacaactcca acgtcaccga cgccatcctc tcccgcgccg gtggttccat cgccgacttc 1200accggtcacc gccagaccgc cttccgcgag ctcgagcgcg tcctcaactt cccccagtcc 1260aacctctgcc tcaagcgcga gaagcaggac gagtcctgct ccctcaccca ggccctcccc 1320tccgagctca aggtctccgc cgacaacgtc tccctcaccg gtgccgtctc cctcgcctcc 1380atgctcaccg agatcttcct cctccagcag gcccagggta tgcccgagcc cggttggggt 1440cgcatcaccg actcccacca gtggaacacc ctcctctccc tccacaacgc ccagttctac 1500ctcctccagc gcacccccga ggtcgcccgc tcccgcgcca cccccctcct cgacctcatc 1560aagaccgccc tcacccccca ccccccccag aagcaggcct acggtgtcac cctccccacc 1620tccgtcctct tcatcgccgg tcacgacacc aacctcgcca acctcggtgg tgccctcgag 1680ctcaactgga ccctccccgg tcagcccgac aacacccccc ccggtggtga gctcgtcttc 1740gagcgctggc gccgcctctc cgacaactcc cagtggatcc aggtctccct cgtcttccag 1800accctccagc agatgcgcga caagaccccc ctctccctca acaccccccc cggtgaggtc 1860aagctcaccc tcgccggttg cgaggagcgc aacgcccagg gtatgtgctc cctcgccggt 1920ttcacccaga tcgtcaacga ggcccgcatc cccgcctgct ccctctaatg ccattggcgg 1980aggggtccgg acggtcagga acttagcctt atgagatgaa tgatggacgt gtctggcctc 2040ggaaaaggat atatggggat catgatagta ctagccatat taatgaaggg catataccac 2100gcgttggacc tgcgttatag cttcccgtta gttatagtac catcgttata ccagccaatc 2160aagtcaccac gcacgaccgg ggacggcgaa tccccgggaa ttgaaagaaa ttgcatccca 2220ggccagtgag gccagcgatt ggccacctct ccaaggcaca gggccattct gcagcgctgg 2280tggattcatc gcaatttccc ccggcccggc ccgacaccgc tataggctgg ttctcccaca 2340ccatcggaga ttcgtcgcct aatgtctcgt ccgttcacaa gctgaagagc ttgaagtggc 2400gagatgtctc tgcaggaatt caagctagat gctaagcgat attgcatggc aatatgtgtt 2460gatgcatg 2468282468DNAArtificial SequenceAmy-PhyM1 expression cassette 28tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc

cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatggtcgcc 660tggtggtccc tcttcctcta cggtctccag gtcgccgccc ccgccctcgc cgccaccccc 720gccgactggc gctcccagtc cgagcccgag ctcaagctcg agtccgtcgt catcgtctcc 780cgccacggtg tccgcgcccc caccaaggcc acccagctca tgcaggacgt cacccccgac 840gcctggccca cctggcccgt caagctcggt gagctcaccc cccgcggtgg tgagctcatc 900gcctacctcg gtcactactg gcgccagcgc ctcgtcgccg acggtctcct ccccaagtgc 960ggttgccccc agtccggtca ggtcgccatc atcgccgacg tcgacgagcg cacccgcaag 1020accggtgagg ccttcgccgc cggtctcgcc cccgactgcg ccatcaccgt ccacacccag 1080gccgacacct cctcccccga ccccctcttc aaccccctca agaccggtgt ctgccagctc 1140gacaacgcca acgtcaccga cgccatcctc gagcgcgccg gtggttccat cgccgacttc 1200accggtcact accagaccgc cttccgcgag ctcgagcgcg tcctcaactt cccccagtcc 1260aacctctgcc tcaagcgcga gaagcaggac gagtcctgct ccctcaccca ggccctcccc 1320tccgagctca aggtctccgc cgactgcgtc tccctcaccg gtgccgtctc cctcgcctcc 1380atgctcaccg agatcttcct cctccagcag gcccagggta tgcccgagcc cggttggggt 1440cgcatcaccg actcccacca gtggaacacc ctcctctccc tccacaacgc ccagttcgac 1500ctcctccagc gcacccccga ggtcgcccgc tcccgcgcca cccccctcct cgacctcatc 1560aagaccgccc tcacccccca ccccccccag aagcaggcct acggtgtcac cctccccacc 1620tccgtcctct tcatcgccgg tcacgacacc aacctcgcca acctcggtgg tgccctcgag 1680ctcaactgga ccctccccgg tcagcccgac aacacccccc ccggtggtga gctcgtcttc 1740gagcgctggc gccgcctctc cgacaactcc cagtggatcc aggtctccct cgtcttccag 1800accctccagc agatgcgcga caagaccccc ctctccctca acaccccccc cggtgaggtc 1860aagctcaccc tcgccggttg cgaggagcgc aacgcccagg gtatgtgctc cctcgccggt 1920ttcacccaga tcgtcaacga ggcccgcatc cccgcctgct ccctctaatg ccattggcgg 1980aggggtccgg acggtcagga acttagcctt atgagatgaa tgatggacgt gtctggcctc 2040ggaaaaggat atatggggat catgatagta ctagccatat taatgaaggg catataccac 2100gcgttggacc tgcgttatag cttcccgtta gttatagtac catcgttata ccagccaatc 2160aagtcaccac gcacgaccgg ggacggcgaa tccccgggaa ttgaaagaaa ttgcatccca 2220ggccagtgag gccagcgatt ggccacctct ccaaggcaca gggccattct gcagcgctgg 2280tggattcatc gcaatttccc ccggcccggc ccgacaccgc tataggctgg ttctcccaca 2340ccatcggaga ttcgtcgcct aatgtctcgt ccgttcacaa gctgaagagc ttgaagtggc 2400gagatgtctc tgcaggaatt caagctagat gctaagcgat attgcatggc aatatgtgtt 2460gatgcatg 2468292468DNAArtificial SequenceAmy-PhyM2 expression cassette 29tgccattggc ggaggggtcc ggacggtcag gaacttagcc ttatgagatg aatgatggac 60gtgtctggcc tcggaaaagg atatatgggg atcatgatag tactagccat attaatgaag 120ggcatatacc acgcgttgga cctgcgttat agcttcccgt tagttatagt accatcgtta 180taccagccaa tcaagtcacc acgcacgacc ggggacggcg aatccccggg aattgaaaga 240aattgcatcc caggccagtg aggccagcga ttggccacct ctccaaggca cagggccatt 300ctgcagcgct ggtggattca tcgcaatttc ccccggcccg gcccgacacc gctataggct 360ggttctccca caccatcgga gattcgtcgc ctaatgtctc gtccgttcac aagctgaaga 420gcttgaagtg gcgagatgtc tctgcaggaa ttcaagctag atgctaagcg atattgcatg 480gcaatatgtg ttgatgcatg tgcttcttcc ttcagcttcc cctcgtgcag atgaggtttg 540gctataaatt gaagtggttg gtcggggttc cgtgaggggc tgaagtgctt cctccctttt 600agacgcaact gagagcctga gcttcatccc cagcatcatt acacctcagc aatggtcgcc 660tggtggtccc tcttcctcta cggtctccag gtcgccgccc ccgccctcgc cgccaccccc 720gccgactggc gctcccagtc cgagcccgag ctcaagctcg agtccgtcgt catcgtctcc 780cgccacggtg tccgcgcccc caccaaggcc acccagctca tgcaggacgt cacccccgac 840gcctggccca cctggcccgt caagctcggt gagctcaccc cccgcggtgg tgagctcatc 900gcctacctcg gtcactactg gcgccagcgc ctcgtcgccg acggtctcct ccccaagtgc 960ggttgccccc agtccggtca ggtcgccatc atcgccgacg tcgacgagcg cacccgcaag 1020accggtgagg ccttcgccgc cggtctcgcc cccgactgcg ccatcaccgt ccacacccag 1080gccgacacct cctcccccga ccccctcttc aaccccctca agaccggtgt ctgccagctc 1140gacaacgcca acgtcaccga cgccatcctc gagcgcgccg gtggttccat cgccgacttc 1200accggtcact accagaccgc cttccgcgag ctcgagcgcg tcctcaactt cccccagtcc 1260aacctctgcc tcaagcgcga gaagcaggac gagtcctgct ccctcaccca ggccctcccc 1320tccgagctca aggtctccgc cgactgcgtc tccctcaccg gtgccgtctc cctcgcctcc 1380atgctcaccg agatcttcct cctccagcag gcccagggta tgcccgagcc cggttggggt 1440cgcatcaccg actcccacca gtggaacacc ctcctctccc tccacaacgc ccagttcgac 1500ctcctccagc gcacccccga ggtcgcccgc tcccgcgcca cccccctcct cgacctcatc 1560aagaccgccc tcacccccca ccccccccag aagcaggcct acggtgtcac cctccccacc 1620tccgtcctct tcatcgccgg tcacgacacc aacctcgcca acctcggtgg tgccctcgag 1680ctcaactgga ccctccccgg tcagcccgac aacacccccc ccggtggtga gctcgtcttc 1740gagcgctggc gccgcctctc cgacaactcc cagtggatcc aggtctccct cgtcttccag 1800accctccagc agatgcgcga caagaccccc ctctccctca acaccccccc cggtgaggtc 1860aagctcaccc tcgccggttg cgaggagcgc aacgcccagg gtatgtgctc cctcgccggt 1920ttcacccaga tcgtcaacga ggcccgcatc cccgcctgct ccctctaatg ccattggcgg 1980aggggtccgg acggtcagga acttagcctt atgagatgaa tgatggacgt gtctggcctc 2040ggaaaaggat atatggggat catgatagta ctagccatat taatgaaggg catataccac 2100gcgttggacc tgcgttatag cttcccgtta gttatagtac catcgttata ccagccaatc 2160aagtcaccac gcacgaccgg ggacggcgaa tccccgggaa ttgaaagaaa ttgcatccca 2220ggccagtgag gccagcgatt ggccacctct ccaaggcaca gggccattct gcagcgctgg 2280tggattcatc gcaatttccc ccggcccggc ccgacaccgc tataggctgg ttctcccaca 2340ccatcggaga ttcgtcgcct aatgtctcgt ccgttcacaa gctgaagagc ttgaagtggc 2400gagatgtctc tgcaggaatt caagctagat gctaagcgat attgcatggc aatatgtgtt 2460gatgcatg 2468

Claims

1. A signal peptide for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus, wherein the signal peptide is derived from Aspergillus oryzae TAKA amylase and has an amino acid sequence as shown in SEQ ID NO. 13.

2. The signal peptide for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 1, having a nucleotide sequence as shown in SEQ ID NO. 12.

3. The signal peptide for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 1, wherein the filamentous fungus is selected from Aspergillus niger.

4. The signal peptide for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 3, wherein the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 4.

5. The signal peptide for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 3, wherein the mutant of the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

6. A codon-optimized gene encoding Escherichia coli phytase or a mutant thereof, which has a nucleotide sequence as shown in SEQ ID NO. 7; or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 7, and encodes a protein having the phytase activity.

7. A codon-optimized DNA sequence encoding Escherichia coli phytase or a mutant mature peptide thereof, which has a nucleotide sequence as shown in SEQ ID NO. 8; or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 8, and encodes a protein having the phytase activity.

8. A method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus, comprising linking the Aspergillus oryzae TAKA amylase signal peptide to a DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof, and inserting an expression cassette comprising the sequence into a filamentous fungus for expression, wherein the nucleotide sequence of the Aspergillus oryzae TAKA amylase signal peptide is as shown in SEQ ID NO. 12.

9. The method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 8, wherein the Aspergillus oryzae TAKA amylase signal peptide has an amino acid sequence as shown in SEQ ID NO. 13.

10. The method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 8, wherein the filamentous fungus is selected from Aspergillus niger.

11. The method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 8, wherein the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 4, and the mutant of the Escherichia coli phytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

12. The method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 8, wherein the DNA sequence encoding Escherichia coli phytase or a mutant mature peptide thereof is not codon optimized.

13. The method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 8, wherein the DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof is codon optimized, and has a nucleotide sequence as shown in SEQ ID NO. 8 or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 8.

14. The method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 13, wherein the codon optimized DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 14.

15. The method for enhancing the secretory expression of Escherichia coli phytase or a mutant thereof in a filamentous fungus according to claim 13, wherein the codon optimized DNA sequence encoding the Escherichia coli phytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 16.